Variants of the major allergen Phl p 1 from timothy grass

ABSTRACT

The invention relates to pharmaceutically important variants of the major allergen Phl p 1 from timothy grass, characterized in that a preparation, hitherto not possible, of monomeric molecules which are soluble and stable in physiological media can be carried out with the aid of prokaryotic expression systems and subsequent purification thereof.

The invention relates to variants of the major allergen Phl p 1 fromtimothy grass, characterised in that a preparation, hitherto notpossible, of monomeric molecules which are soluble and stable inphysiological media can be carried out with the aid of prokaryoticexpression systems and subsequent purification thereof.

BACKGROUND OF THE INVENTION

Type 1 allergies are of worldwide importance. Up to 25% of thepopulation of industrialised countries suffer from complaints such asallergic rhinitis, conjunctivitis or bronchial asthma which are causedby allergens of various origin present in the air (aeroallergens), suchas plant pollen, mites, cats or dogs. Up to 40% of these type 1 allergysufferers in turn exhibit specific IgE (immunoglobulin E) reactivity inthe case of grass pollen (Freidhoff et al., 1986, J. Allergy. Clin.Immunol. 78, 1190-201).

The substances which initiate type 1 allergy are proteins, glycoproteinsor polypeptides. After uptake via the mucous membranes, these allergensreact with the IgE molecules bonded to the surface of mast cells insensitised people. If two IgE molecules are crosslinked to one anotherby an allergen, this results in the release of mediators (for examplehistamine, prostaglandins) and cytokines by the effector cell and thusin the corresponding clinical symptoms.

Depending on the relative frequency of the allergy sufferers having IgEantibodies against certain allergens, a distinction is made betweenmajor and minor allergens. In the case of timothy grass (Phleumpratense), Phl p 1 (Petersen et al., 1993, J. Allergy Clin. Immunol. 92,789-796), Phl p 5 (Matthiesen and Löwenstein, 1991, Clin. Exp. Allergy21, 297-307), Phl p 6 (Petersen et al., 1995, Int. Arch. AllergyImmunol. 108, 49-54) and Phl p 2/3 (Dolecek et al., 1993) have hithertobeen characterised as major allergens and Phl p 4 (Löwenstein, 1978,Prog. Allergy 25, 1-62) and group 10 and 11 from Lolium perenne (Ansariet. al., 1987, J. Allergy Clin. Immunol. 80, 229-235) as minorallergens.

Group 1, which includes Phi p 1 from timothy grass, is classified as oneof the most relevant allergen groups of grass pollen (Tamborini, E. etal., Eur. J. Biochem. 1997, 249:886-894). The other representatives ofgroup 1 from other grasses have homologies of in some cases more than95% to Phl p 1 (Petersen, A., et al., J. Allergy Clin. Immunol. 1995,95: 987-994). Owing to the high homologies, reactions to the allergensof other cross-reactive species also occur in the case of sensitisationwith a grass. For this reason, these molecules are of overridingimportance for corresponding diagnostic and therapeutic approaches.

On therapeutic use of these allergens, use is made of the reaction withT helper cells, where reorientation of the pathological TH2 cells intothe TH1 type occurs. This causes a change in the cytokine profile sothat B cells are stimulated to form IgG instead of IgE.

A classical approach to effective therapeutic treatment of allergies isspecific immunotherapy or hyposensitisation (Fiebig, 1995, Allergo J. 4(6), 336-339, Bousquet et al., 1998, J. Allergy Clin Immunol. 102(4),558-562), in which the patient is injected subcutaneously with naturalallergen extracts in increasing doses.

However, there is a risk in this method of allergic reactions or evenanaphylactic shock. In order to minimise these risks, innovativepreparations in the form of allergoids are being employed. These arechemically modified allergen extracts which have significantly reducedIgE reactivity, but identical T-cell reactivity compared with theuntreated extract (Fiebig, 1995, Allergo J. 4 (7), 377-382).

Even more substantial therapy optimisation would be possible withallergens prepared by recombinant methods. Defined cocktails, optionallymatched to individual patients, of highly pure allergens prepared byrecombinant methods could release extracts from natural allergen sourcessince these, in addition to the various allergens, contain a relativelylarge number of immunogenic, but non-allergenic accompanying proteins.Realistic perspectives which may result in reliable hyposensitisationwith expression products are offered by specifically mutated recombinantallergens in which IgE epitopes are specifically deleted withoutimpairing the T-cell epitopes which are essential for therapy (Schrammet al., 1999, J. Immunol. 162, 2406-2414).

A further possibility for therapeutic influencing of the disturbedTh-cell equilibrium in allergy sufferers is treatment with expressibleDNA which encodes for the relevant allergens. Initial experimentalevidence of allergen-specific influencing of the immune response hasbeen furnished in rodents by ejection of allergen-encoding DNA (Hsu etal., 1996, Nature Medicine 2 (5), 540-544).

Phl p 1 is a protein comprising 240 amino acids and an n-glycosylationsite. The glycosylation fraction is 5% of the molecular weight, which,in the natural protein, is about 30-35 kDa (Petersen et al., AllergyClin. Immunol. 1995, 95: 987-994; Suck et al., J. Immunol. Meth. 1999,229:73-80). The nucleic acid sequence of Phl p 1 is known (Laffer etal., J. Allergy Clin Immunol. 1994, 94: 689-698; Petersen et. al., J.Allergy Clin. Immunol., 1995, 95: 987-94) and can thus be utilised forthe recombinant preparation of the molecule.

Previous attempts to prepare the molecule by recombinant methods inbacterial or eukaryotic systems, such as, for example, yeast, in such away that a stable monomeric form was obtained were, however,unsuccessful owing to its poor solubility:

In the case of bacterial expression, Phl p 1 is deposited as inclusionbodies (Vrtala et al., J. Allergy Clin. Immunol, 1996; 97: 781-7) andfirstly has to be denatured before purification. The denaturing agent issubsesequently removed. However, complete re-folding of the protein intothe natural soluble conformation has hitherto not been achieved(Andersson, Lidholm, Int. Arch. Allergy Immunol. 2003; 130:87-107).

One possible reason preventing the formation of a stable conformationcould have been the absence of glycosylation. However, a stable Phl p 1has not been obtained even in eukaryotic systems in which glycosylationis possible (K. Grobe, Dissertation, 1998, University of Hamburg).

Instead, a cause of the lack of solubility is assumed to be proteolyticactivity, which results in self-degradation of the molecule (Grobe etal., Eur. J. Biochem. 1999; 263: 3340; Kirsten Gehlhar, Dissertation,1998, Medical University of Lübeck, Germany). In addition, hydrophobicinteractions between the molecules are also possible as a cause of theaggregation.

The object on which the present invention is based thus consisted in theprovision of variants of the major allergen Phl p 1 from timothy grasswhich are distinguished by improved solubility at the same time as fullretention of the therapeutically and diagnostically importantimmunological properties and which can thus be purified inpharmaceutically suitable form.

FIGURES

FIG. 1: SDS-PAGE of wild type nPhl p 1 (n=natural) and fold variants LMand HM of allergen variant rPhl p 1-A236C (r=recombinant) under reducingconditions (in the presence of dithiothreitol DTT) and non-reducingconditions (without DTT).

-   Track 1: Molecular weight standard (10, 15, 20, 25, 30, 40, 50, 60,    70, 80, 90, 100, 120, 160, 220 kDa, bench mark protein ladder,    Invitrogen, Karlsruhe, Germany)-   Track 2: Extract from Phleum pratense pollen-   Track 3: nPhl p 1-   Track 4: rPhl p 1-LM-   Track 5: rPhl p 1-HM

FIG. 2: Gel filtration with rPhl p 1-HM and rPhl p 1-LM on a SephacrylS100 column. The figure shows that the two fold variants have differentapparent molecular weights.

FIG. 3: Enzyme allergo-sorbent test (EAST) for quantification of the IgEbonding of fold variants rPhl p 1-A236C -LM and -HM The concentration ofan inhibitor of IgE-nPhl p 1 bonding in mol/l is plotted on thehorizontal axis, the degree of inhibition in [%] is indicated on thevertical axis. The measurement was carried out with nPhl p 1 on thesolid phase and a typical serum of a grass pollen allergy sufferer.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has now been found that the introduction of anadditional cysteine residue, preferably in the carboxyl-terminated part(in particular from amino acid position 140, very particularlypreferably between amino acid positions 230 and 240) of the molecule,results in the improved solubility in accordance with the invention withunchanged IgE activity and T-cell reactivity.

The invention therefore relates to variants of the major allergen Phl p1 from timothy grass which have an additional Cys residue compared withthe wild type, and to fragments and variants derived from the basemolecules which have the same or similar advantageous properties.

The invention furthermore relates to a process for the preparation ofthe variants according to the invention of the recombinant majorallergen rPhl p 1, characterised in that, by methods known per se, abase triplet encoding for a Cys residue is introduced into the Phl p 1gene by insertion or exchange, the gene modified in this way isoverexpressed in a host organism, and the allergen variant obtained byoverexpression is purified.

The prokaryotic recombinant preparation and purification can be carriedout with or without a fusion component introduced by genetic engineeringand always results in the same products. If a fusion component is used,it is preferably an His tag. The purification methods vary depending onthe expression vector or system.

The present invention accordingly encompasses a specifically modifiedprimary sequence of the recombinant allergen rPhl p 1 which facilitatesthe recombinant preparation thereof in bacterial or other expressionsystems and subsequent purification.

The invention thus also relates to DNA molecules which encode for theallergen variants according to the invention.

The recombinant proteins are autoproteolytically inactive and cantherefore be stored in stable monomeric form in physiological, bufferedor other solutions depending on the application. The T-cell stimulationexhibits no significant differences between recombinant and natural Phlp 1.

The recombinant allergen variants and the derived fragments or variantscan thus be utilised for the therapy of grass pollen-induced allergicdiseases.

Owing to this pharmaceutical suitability, the present invention alsorelates to the novel allergen variants in their property as medicaments.

The allergen variants and fragments prepared by recombinant methods canfurthermore be utilised for the diagnosis of pollen allergies.

In the preparation of the major allergen Phl p 1 from timothy grassmodified by genetic engineering, the amino acid exchange is effected bytargeted nucleotide exchange, for example by means of PCR. In aparticularly preferred embodiment, the mutant rPhl p 1-A236C inaccordance with SEQ ID NO 2, the alanine at position 236 is replaced bycysteine. However, the exchange site can also be at any desired othersite of the molecule. In general, however, it will be localised in theC-terminal region of the molecule, preferably from position 140, inparticular between positions 230 and 240.

As a consequence of the exchange, the proteolytic activity known for Phlp 1 is simultaneously eliminated.

As a further unexpected effect, two fold variants which can becompletely separated from one another occur during the isolation andpurification of the molecule in accordance with the invention.

While the first conformation variant, referred to as rPhl p 1-LM (LM=lowmolecular weight), exhibits very similar behaviour to the naturalprotein owing to its similar or identical run behaviour in thenon-reduced SDS-PAGE (FIG. 1 ) and gel filtration (for example onSepharcryl S-100, cf. FIG. 2), the second variant, referred to as rPhl p1-HM (HM=high molecular weight), exists in a different fold form. TheIgE reactivity also differs. While rPhl p 1-LM has a reactivitycomparable to the natural protein, rPhl p 1-HM is bound less well by IgEantibodies and is particularly suitable for specific immunotherapy owingto its hypoallergeneity (see FIG. 3).

In principle, however, both fold forms are suitable both for therapeuticand for diagnostic applications.

The invention thus furthermore relates to different fold forms of therPhl p 1 allergen variant according to the invention and to the usethereof for therapeutic and diagnostic purposes.

Both fold forms are readily soluble, stable in monomeric form and haveno detectable proteolytic/autoproteolytic activity.

The allergen variants according to the invention can be obtained, forexample, by the two preparation processes outlined below—with or withoutartificial fusion component:

1) Expression with Artificial Fusion Component (His tag)

In the case of the use of an His tag, the purification of the initiallyinsoluble crude protein is carried out via a plurality of biochemicalseparation steps, comprising one or more metal ion chelate affinitychromatography steps and removal of the His tag. For pre- andpost-purification, various other chromatography methods and de- andrenaturing steps can be used.

Preparation of Fold Form rPhl p 1-LM:

-   -   denaturing of the inclusion bodies isolated from the host        organism using guanidinium chloride,    -   renaturing of the dissolved protein on a chelate affinity        chromatography column,    -   removal of the His tag,    -   first gel filtration,    -   chelate affinity chromatography,    -   isolation of the target protein from the flow-through, second,        final gel filtration.

The other fold form—rPhl p 1-HM—can be obtained in this purificationvariant by carrying out the following process steps:

-   -   denaturing of the inclusion bodies isolated from the host        organism using guanidinium chloride,    -   renaturing of the dissolved protein on a chelate affinity        chromatography column,    -   removal of the His tag,    -   first gel filtration,    -   chelate affinity chromatography,    -   elution of the target protein with an imidazole gradient,    -   second, final gel filtration.        2) Expression Without Fusion Component (Without His tag)    -   Denaturing of the inclusion bodies isolated from the host        organism using guanidinium chloride, where, as described below,        one or other fold form is obtained depending on the denaturing        duration.    -   Renaturing of the dissolved protein by dilution in buffer        solution, where 20-50 mM Tris/HCl pH 7-8 is preferably used, but        other buffers and pH values (for example 7-10.5) are also        possible. For dilution, the denaturing batch is preferably added        to a multiple of its volume (about 10 to 80 times, preferably 20        to 60 times the volume) of the preferably stirred or otherwise        mixed buffer solution—for example by decantation, pipetting or        pumping; however, the buffer solution can also be added to the        denaturing batch. The rate of addition is not crucial: thus, the        entire amount can be added in one portion within a few seconds        or alternatively—(but preferably not necessarily)        uniformly—distributed over a number of hours.    -   Concentration and purification of the renatured protein by        chelate affinity chromatography and subsequent elution with an        imidazole gradient (use is preferably made of a step gradient,        but a continuous gradient is likewise possible). Alternatively        or additionally to the chelate affinity chromatography,        hydrophobic interaction chromatography and/or an anion exchange        chromatography can optionally also be carried out (the molecules        must be treated completely by chromatography).    -   Final gel filtration.

The alternative stable fold forms LM and HM can be obtained specificallyhere with minimal cross-contamination by means of different incubationtimes in the denaturing step. For the preparation of the LM form,incubation can basically be carried out for between about 1 and 50hours. In general, however, a range from 10 to 40 hours, preferably from15 to 30 hours, will be used, where a range from 18 to 22 hours isparticularly preferred.

For the HM variant, significantly longer incubation times are required.They are in the order of from 60 to 120 hours. However, incubation willusually be carried out for from 70 to 100 hours, particularly preferablyin a range of from 80 to 90 hours.

The denaturing step is in all cases followed by the above-describeddilution step with buffer solution.

The interval between (about 50 to 60 hours) represents the re-formationphase. The dilution step apparently fixes the folding process, nofurther kinetics take place.

The fine purification for the separation of LM and HM is possible viahydrophobic interaction chromatography or is achieved by gel filtration,in which the forms have significantly different retention times.

The allergen variants according to the invention can be obtained in highpurity in their fold variants LM and HM and have valuablepharmaceutically relevant properties. Thus, they can be employed as amixture, but also individually, for diagnosis (in particular rPhl p 1-LMowing to the retention of IgE activity) and therapy (in particular rPhlp 1-HM owing to the reduced IgE activity) of allergic diseases.

The invention therefore likewise relates to the use of the allergenvariants and/or pharmaceutically usable derivatives thereof, includingmixtures thereof in all ratios, for the preparation of a medicament forspecific immunotherapy (hyposensitisation) and diagnosis of allergies inthe triggering of which the major allergen Phl p 1 from timothy grass isinvolved.

The invention furthermore relates to a pharmaceutical compositioncomprising an allergen variant according to the invention and/orpharmaceutically usable derivatives thereof, including mixtures thereofin all ratios, and, if desired, excipients and/or adjuvants. The activeingredients according to the invention can be converted into a suitabledosage form here together with at least one solid, liquid and/orsemi-liquid excipient or adjuvant and, if desired, in combination withone or more further active ingredients.

If the DNA molecules on which the allergen variants according to theinvention are based are ligated to a suitable expression vector, theseconstructs can in addition be used as preparations for immunotherapy(DNA vaccination).

The invention therefore likewise relates to a recombinant DNA expressionvector containing a DNA molecule according to the invention for thetreatment of allergies in the triggering of which the major allergen Phlp 1 from timothy grass is involved, by immunotherapeutic DNAvaccination.

The invention furthermore relates to the use of the said expressionvector and/or derivatives thereof, including mixtures thereof in allratios, for the preparation of a medicament for the treatment ofallergies in the triggering of which the major allergen Phl p 1 fromtimothy grass is involved, by immunotherapeutic DNA vaccination.

Finally, the invention relates to a pharmaceutical compositioncomprising the said expression vector and/or pharmaceutically usablederivatives thereof, including mixtures thereof in all ratios, and, ifdesired, excipients and/or adjuvants, for the treatment of allergies inthe triggering of which the major allergen Phl p 1 from timothy grass isinvolved, by immunotherapeutic DNA vaccination.

For the purposes of this invention, pharmaceutical compositions can beused as therapeutic agents in human or veterinary medicine. Suitableexcipients are organic or inorganic substances which are suitable forparenteral administration and do not react with allergen variantsaccording to the invention of the major allergen Phl p 1 from timothygrass. Suitable for parenteral administration are, in particular,solutions, preferably oil-based or aqueous solutions, furthermoresuspensions, emulsions or implants. The allergen variants according tothe invention may also be lyophilised and the resultant lyophilisatesused, for example, for the preparation of injection preparations. Thecompositions indicated may be sterilised and/or comprise adjuvants, suchas lubricants, preservatives, stabilisers and/or wetting agents,emulsifiers, salts for modifying the osmotic pressure, buffer substancesand/or a plurality of further active ingredients.

Furthermore, delayed-release compositions can be obtained bycorresponding formulation of the allergen variants according to theinvention, for example by adsorption on aluminium hydroxide.

Further selective modifications at different positions and othermodifications—for example for increasing the hypoallergeneity—are ofcourse also possible via the mutations according to the invention. Thesemodifications can be, for example, chemical modifications of theallergen extract (Fiebig, 1995, Allergo J. 4 (7), 377-382). However,they can also be carried out at the DNA level by genetic engineering,where, for example, amino acid insertions, deletions and exchanges,cleavage of the protein into fragments and fusion of the protein orfragments thereof to other proteins or peptides are suitable.

In view of the high sequence homologies within group 1 grass pollenmajor allergens, all the effects described here for Phl p 1 relating tothe improvement in the solubility by the introduction of a Cis residueand the occurrence of fold variants can also be expected for otherrepresentatives of this group.

Even without further comments, it is assumed that a person skilled inthe art will be able to utilise the above description in its broadestscope. The embodiments depicted below in Tables 1 and 2 by way ofexample for the variant rPhl p 1-A236C should therefore merely beregarded as descriptive disclosure which is absolutely not limiting inany way.

All chromatography materials are commercially available from AmershamBiosciences (Freiburg, Germany).

The choice of metal ion in the chelate affinity chromatography methodsdescribed is not crucial, both Ni and Cu can be used.

EXAMPLE 1 Isolation of rPhl p 1-A236C-LM and -HM—Purification Variantwith His tag

The sequence encoding for Phl p 1 was amplified by means of PCR with 5′-and 3′-specific oligonucleotides and ligated into a pProEx vector(GIBCO, La Jolla, USA) via the Ehe I and Hind III restriction site. The3′-primer was modified from GC to TG at base position 706/707 in such away that an alanine-encoding triplet is converted into acysteine-encoding triplet (Essential Molecular Biology; T. A. Brown ed.,IRL Press, Oxford, 1994). The transformation was carried out in E. coliorigami. The starting vector pProEx selected supplies the 6×His sequencelocalised at the N-terminal end, followed by a recognition sequence forthe TEV protease. The recombinant, primarily 6×HIS-tagged rPhl p 1-A236Cmolecules present as insoluble aggregates after bacterial expression aredissolved after pre-purification in 6 M guanidinium chloride (GdmCl), 50mM Tris/HCl, 500 mM NaCl, pH 8.0). This is followed by two-stage Nichelate affinity chromatography:

In a first step, the proteins bonded to chelating Sepharose underdenaturing conditions are transferred by a gradient over the course of90 min from the denaturing solution into a buffer consisting of 50 mMphosphate buffer and 500 mM NaCl (pH 7.4). This is followed by stepwiseelution with 500 mM imidazole in phosphate buffer. The renatured fusionprotein is cleaved into rPhl p 1 and the 6×His fusion component by meansof a specific TEV protease.

For preparation for a second affinity chromatography, a gel filtrationis carried out with Sephadex G-25 and phosphate buffer as eluent, as aresult of which the imidazole is removed.

The rebuffered protein mixture is then employed for a second Ni chelateaffinity chromatography. In this, some of the successfully cleaved rPhlp 1 is found in the flow-through. This is the LM form of the molecule.Besides uncleaved molecules, the conformation variant HM also remainsadhering to the column, apparently due to exposed histidine residues.This cleaved variant elutes before the uncleaved fusion proteins in animidazole gradient, enabling this form also to be obtained in highpurity. In a final step, a gel filtration with Superdex 75 is carriedout for final purification and transfer into a desired solvent.

TABLE 1 Overview of the preparation and purification process accordingto the invention using an His tag 1. Expression 2. Isolation of theinclusion bodies 3. Denaturing 4. Ni chelate affinity chromatography 1(renaturing) 5. Removal of the His tag 6. Gel filtration (Sephadex G-25)7. Ni chelate affinity chromatography 2: Flow-through: rPhl p 1-LMEluate: rPhl p 1-HM, Uncleaved His-rPhl p 1 fusion protein 8. Gelfiltration (Superdex 75)

EXAMPLE 2 Isolation of rPhl p 1-A236C-LM and -HM—Purification Variantwithout His tag

Initially, the procedure in accordance with Example 1 is followed, butwhere the vector selected does not supply a fusion protein as primaryproduct.

The recombinant, primarily rPhl p 1-A236C molecules present as insolubleaggregates (inclusion bodies) after bacterial expression, are dissolvedafter pre-purification in 6M guanidinium chloride (GdmCl), 50 mMTris/HCl, pH 8.0). This is followed by 40-fold dilution in 20 mM Tris pH8.0. To this end, the denaturing solution is decanted into the dilutionsolution stirred using a magnetic stirrer.

a) Preparation of the LM Form

The inclusion bodies are incubated in the denaturing solution for 20 hand subsequently diluted as described above.

b) Preparation of the HM Form

The inclusion bodies are incubated in the denaturing solution for 85 hand subsequently diluted as described above.

500 mM NaCl are added to the respective dilution (renaturing) batch. Thedissolved molecules in the renaturing batch are concentrated via Cuchelate affinity chromatography and eluted with 200 mM imidazole inphosphate buffer as stage (or gradually through 500 mM imidazole inphosphate buffer).

The chelate affinity chromatography can optionally be utilised forconditioning before elution of the protein by, for example, using a 3 MNaCl solution for washing and a 3 M NaCl, 200 mM imidazole solution forthe elution. The eluate, which has a high salt content, could then beemployed directly for hydrophobic interaction chromatography.

In a final step, a gel filtration with Superdex 75 is carried out forfinal purification and transfer into a desired solvent.

TABLE 2 Overview of the preparation and purification process accordingto the invention without use of an His tag 1. Expression 2. Isolation ofthe inclusion bodies 3. Denaturing Duration 20 h: LM Duration 85 h: HM4. Dilution 5. Cu chelate affinity chromatography 6. Gel filtration(Superdex 75)

EXAMPLE 2 Different IgE Bonds of the Wild Type and of Fold Variants LMand HM of Allergen Variant rPhl p 1-A236C

Natural nPhl p 1 and recombinant rPhl p 1 variants HM and LM arecompared with one another with respect to the strength of their IgEbonding in an EAST inhibition assay carried out by the method of Suck etal. (Int. Arch. Allergy Immunol. 2000; 121: 284-291) with an allergysufferer serum pool (FIG. 3). It is found that the HM variant issignificantly reduced in its IgE bonding compared with natural Phl p 1protein, while the LM variant has IgE bonding which is comparable tonatural Phl p 1 protein.

1. An isolated polypeptide variant of major allergen Phl p 1 fromtimothy grass which comprises the polypeptide sequence set forth in SEQID NO: 2, wherein the variation comprises an additional Cys residue thathas been introduced by exchange of Ala 236 in the wild-type Phl p 1polypeptide sequence.
 2. The isolated polypeptide variant of claim 1which is an LM or HM fold variant of said polypeptide of SEQ ID NO: 2.3. A fold form rPhl p 1-LM of the isolated polypeptide variant accordingto claim 1, which is obtainable by: (a) overexpressing in a hostorganism, a fusion protein comprising the rPhl p 1 polypeptide variantand a His tag; (b) denaturing inclusion bodies isolated from the hostorganism using guanidinium chloride; (c) renaturing dissolved protein ona chelate affinity chromatography column; (d) removing the His tag; (e)employing gel filtration; (f) further purifying using chelate affinitychromatography; (g) isolating the target protein from the flow-through;and (h) further employing gel filtration.
 4. A fold form rPhl p 1-HM ofthe isolated polypeptide variant according to claim 1, which isobtainable by: (a) overexpressing in a host organism, a fusion proteincomprising the rPhl p 1 polypeptide variant and a His tag; (b)denaturing inclusion bodies isolated from the host organism usingguanidinium chloride; (c) renaturing dissolved protein on a chelateaffinity chromatography column; (d) removing the His tag; (e) employinggel filtration; (f) further purifying using chelate affinitychromatography; (g) eluting the target protein with an imidazolegradient; and (h) further employing gel filtration.
 5. A pharmaceuticalcomposition comprising an isolated polypeptide variant according toclaim 1 and a pharmaceutically acceptable carrier.
 6. The isolatedpolypeptide variant of major allergen Phl p 1 from timothy grassaccording to claim 1 which is (a) a polypeptide which is encoded by apolynucleotide consisting of the sequence set forth in SEQ ID NO: 1; or(b) a polypeptide which consists of the sequence set forth in SEQ ID NO:2.